The outcome of hyperthermia treatments at the molecular level is governed by antagonism between the tendency of elevated temperatures to cause unfolding and lethal aggregation of cellular proteins and the protection of cells from the consequences of protein denaturation by the induction of the highly conserved heat shock response. The heat shock response is an efficient homeostatic mechanism regulated both at the posttranslational level and by transcriptional activation of the heat shock genes. In this project, we aim to explore the regulatory and signal transduction mechanisms that control and coordinate the heat shock response. We will investigate the role of heat shock proteins (HSPs) as feedback regulators of the heat shock response that function by sequestering mediators of the response such as the heat shock transcription factor (HSF-I) in protein complexes. We will test the hypothesis that constitutive HSPs repress the response under resting conditions, hyperthermia relieves the repression and that inducible HSPs shut down the response during recovery. We will study regulatory HSP complexes, their behavior under heat shock and recovery conditions and thus assess their potential role as negative regulators of the response. Although the reversal of negative regulation may be necessary for the activation of heat shock response it is apparently not sufficient and positive signals emanating from protein kinase cascades are required. We aim to link these cascade responses to upstream signals generated at the plasma membrane. We will concentrate on examining the activity of the ERK family of protein kinases that are powerfully activated by hyperthermia, exploring the upstream inducing events and examining the downstream targets of this protein kinase family. Hyperthermia also generates calcium signals that appear to control several important aspects of the heat shock response such as the conversion of HSF- l to a DNA binding and nuclear localized form during heat shock and the interaction of HSP7O proteins with the regulatory protein calmodulin. We will examine the role of calcium and calmodulin in the heat shock response and the influence of these cellular regulators on the protein- protein interactions and kinase cascades that govern cellular responses to hyperthermia. Our emphasis throughout will be on determining how these regulatory mechanisms are coordinated to provide a tightly controlled but highly temperature sensitive response that leads to thermotolerance and ultimately to plan pharmacological approaches aimed at antagonizing the effects of thermotolerance.